| Ghrelin is a polypeptide initially found in rat stomach in 1999 by Kojima. It was discovered as the endogenous ligand of growth hormone secretagague receptor(GHSR) and so named because it could stimulate the release of growth hormone (GH). It was reported to have many important physiological roles mediated by its receptors in the release of GH, energy metabolism, secretion of gastric acid, cardiovascular functions, sleep, immunity, cell multiplication, reproduction and so on. At present the studies on ghrelin have been focused on human, mouse, rat, sheep, pig, chicken and so on, but there is little research of ghrelin in rabbit. Therefore we study in detail the gene cloning and distribution of ghrelin in rabbit by immunohistochemistry and RT-PCR. The result is as follows:1. The immunohistochemical SP method was used to study the distribution of ghrelin immunoreactive cells in rabbit gastrointestinal tract. In the stomach, most of ghrelin immunoreactive cells were observed in the base to neck of the fundic gland. A few ghrelin cells were observed in the oxyntic gland and cardiac gland regions. In the duodenum, jejunum, ileum, cecum and colon, ghrelin immunoreactive cells were scattered in the epithelia of the crypts and villi. The ghrelin immunoreactive cells were classed into two cell types, opened-type cells and closed-type cells. Opened-type cells were triangular with their apical cytoplasmic process in contact with lumen. But closed-type cells were round and oval in shape with no contact with lumen. The density of ghrelin immunoreactive cells was decresead from the stomach to colon with the highest in the stomach. The number of the opened-type cells gradually increased in the direction from stomach to the lower gastrointestinal tract.2. The distribution of ghrelin immunoreactive neurons in rabbit CNS was studied by immunohistochemical SP method. The results showed that ghrelin-containing neurons were detected in the hypothalamus, cerebral cortex, medulla and spinal cord. In the hypothalamus, ghrelin-containing neurons were identified in the arcuate, paraventricular, dorsomedial and ventromedial hypothalamic nuclei, and lateral hypothalamic area. Ghrelin-containing neurons were mainly distributed in the lay V of the cerebral cortex, the dorsal motor nucleus of the vagus nerve in the medulla oblongata and the ventral horn in the spinal cord.3. The cloning and distribution of ghrelin mRNA in rabbit were studied by RT-PCR. RNA was extracted from the rabbit stomach and the primer was designed according to the sequence of pig ghrelin mRNA in Genbank. Ghrelin gene was amplified by RT-PCR, the PCR fragrnens were cloned into the pMD18-T vector, then transformed into DH 5a competent cell. The positive plasmid was sequenced and PCR product was analysed to have a 326 bp coding part of ghrelin. The sequence of the product was confirmed to be that of ghrein in rabbit and enrolled in GenBank(accession number: EF566009). The distribution of ghrelin mRNA was studied by RT-PCR. The results revealed the distributions of ghrelin mRNA were extensive, including the cerebrum, hypothalamus, pituitary, brain stem, spinal cord, stomach, duodenum, jejunum, ileum, cecum, heart, liver, spleen, lung, kidney, testicle, ovary and submaxillary gland. The results in this study were similar to those obtained in human and other animals, indicating the conservative of ghrelin in phylogeny interspecies. The present study provided basic information for further studies on ghrelin physiological functions.
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